446

LYSOPHOSPHOLIPASE

[42]

[42] P h o s p h o l i p a s e B f r o m P e n i c i l l i u m n o t a t u m B y KUNIHIKO SAITO, JUNKO SUGATANI,

and TADAYOSHI OKUMURA

Introduction A pure enzyme isolated from aqueous extracts of Penicillium notatum is a glycoprotein with a molecular weight of about 95,000 that catalyzes reactions (1) and (2). The enzymatic activity concerned with reaction (1) is phospholipase B activity (B activity), as proposed by McMurry and Magee, ~and that concerned with reaction (2) is lysophospholipase activity (lyso activity). 1,2-Diacyl-sn-glycero-3-phosphocholine--~ fatty acids + glycerophosphocholine 1-(or 2-)Acyllysoglycerophosphocholine --> fatty acid + glycerophosphocholine

(1) (2)

We propose that the active glycoprotein for phospholipase B has two phospholipase activities. When phospholipase B undergoes limited proteolysis by endogenous protease(s) in P. notatum, which often occurs at the initial stage of purification, the B activity decreases greatly, but all of the lyso activity remains. 2 In this chapter, the original phospholipase B is described as the native form of phospholipase B and the partially proteolyzed one considered as the modified form. Similar phospholipases have been reported by others in microorganisms, plants, and animal tissues, some of which are described under different names, for example, calcium-independent phospholipase A 2 activity (A 2 activity), 3 lysophospholipase with phospholipase A~ activity (A~ activity), or a kind of phospholipase B.4 Recent papers describe phospholipase B from Torulaspora deibrueckii, 5 Saccharomyces cerevisiae, 6 and intestinal brush border membranes.7 However, the correlation between B activity and lyso activity on a protein chemistry level has not been reported. On the other hand, a lysophospholipase which does not catalyze the deacylation of diacylglycerophospholipids but only of monoacylglycero1 W. C. McMurray and W. L. Magee, Annu. Rev. Biochem. 41, 129 (1972). 2 j. Sugatani, T. Okumura, K. Saito, K. Ikeda, and K. Hamaguchi, J. Biochem. (Tokyo) 95, 1407 (1984). 3 S. Pind and A. Kuksis, Biochim. Biophys. Acta 901, 78 (1987). 4 y. Nishijima, Y. Akamatsu, and S. Nojima, J. Biol. Chem. 249, 568 (1974). s y. Kuwabara, M. Maruyama, Y. Watanabe, and S. Tanaka, J. Biochem. (Tokyo) 104, 236 0988). 6 W. Witt, M. E. Schweingruber, and A. Mertshing, Biochim. Biophys. Acta 795, 108 (1984). A. Gassama-Diane, J. Fanvelt, and H. Chap, J. Biol. Chem. 264, 9470 (1989).

METHODS IN ENZYMOLOGY, VOL. 197

Copyright © 1991 by Academic Press, Inc. All fights of reproduction in any form reserved.

[42]

PHOSPHOLIPASE B FROMPenicillium notatum

447

phospholipids has been reported: But it is still uncertain whether the lysophospholipase is an artifact of the phospholipase B produced by limited proteolysis as seen in P. notatum.

Assay Systems Phospholipase B Activity Standard System. The substrate (1-2 gmol) is dispersed in 0.4 ml of 0.1 M acetate buffer (pH 5.0) containing 2 mM EDTA; 0.1 ml of enzyme solution (1-4/zg protein) is added, and the mixture is incubated for various times at 30°. The substrate (3-4 mM) is dispersed in water ultrasonically using a Branson sonicator with a titanium probe at an output of 60 W for 10 min in an ice-salt bath. The titanium particles produced during dispersion are removed by filtration with a Millipore filter. After incubation, the reaction is stopped by adding 0.1 ml of 5% bovine serum albumin (BSA) followed by 0.4 ml of 10% perchloric acid on ice. 9 After vortexing, the mixture is centrifuged, and 1.0 ml of the supernatant is assayed for glycerophosphate esters by the methods of Wells and Dittmer l° or Bartlett.~l Radioactive Substrates. The assay system is the same as the above except for the use of radioactive substrates. After various incubation times, the enzyme reaction is stopped by adding 1.0 ml methanol and 0.5 ml chloroform (one-phase system of Bligh and Dyerl2), followed by a further 0.5 ml of chloroform and water. After centrifugation, the chloroform layer is removed and washed once with 1.0 ml of methanol-water (10 : 9, v/v); the combined methanol-water layers are washed with chloroform. Each of the combined chloroform and methanol-water phases is counted for 14Cand 3zp in a scintillation counter. Aliquots of the chloroform phase are then subjected to thin-layer chromatography (TLC) on silica gel H in the solvent system of chloroform-methanol-acetic acid-water (25 : 15 : 4 : 2, by volume). Radioactive spots on the TLC plates located by autoradiography are scraped directly into counting vials, and 32p and ~4C activities are determined. The methanol-water phase of the hydrolyzate is chromatographed on Whatman No. I paper in n-butanol-acetic acid-water (5 : 3 : 1, by volume). Radioactive spots are located by autoradiography, cut out and counted directly. Results show that 32p_ and 14C-labeled 1,2-diacyl-sn-glycero-3-

8 H. van den Bosch and J. G. De Jong, Biochim. Biophys. Acta 398, 244 (1975). 9 R. M. C. Dawson, Biochem. J. 70, 559 (1958). l0 M. A. Wells and J. C. Dittmer, Biochemistry 5, 3405 (1966). 11D. R. Bartlett, J. Biol. Chem. 234, 466 (1959). 12E. C. Blighand W. F. Dyer, Can. J. Biochem. Physiol. 37, 911 (1959).

448

LYSOPHOSPHOLIPASE

[42]

100

(S2p,14C) GPC A

114C) Fatty Acid

O ~r

t~

O.

'5

50

I

I

,14C) PC

"C

( :~"P,14C) lyso-PC 10

,~(A v 20 30

' 40

, 50

m, 60

Time (min)

FIG. 1. Hydrolysisof 32p. and 14C-labeled1,2-diacyl-sn-glycero-3-phosphocholine.GPC, Glycerophosphocholine;lyso-PC,l-(or 2-)acyl-sn-glycero-3-phosphocholine;PC, 1,2-diacylsn-glycero-3-phosphocholine. phosphocholine is hydrolyzed with the concomitant release of ~4C-labeled fatty acids and [14C,32p]glycerophosphocholine (Fig. 1). No labeled lyso compound is detected. Throughout the reaction, only one radioactive spot is determined on chromatograms of the water-soluble hydrolysis products, and it has the same Rf value as authentic glycerophosphocholine. Assay in Presence of Detergent. In the presence of Triton X-100, the system consists of 5 mM substrate, 0.2 M acetate buffer (pH 5.0) containing 2 mM EDTA, 30 mM Triton X-100, and the enzyme protein in a final volume of 0.4 ml. After incubation, 1.5 ml of a chloroform-methanol mixture (2 : 1, v/v) is added, followed by 0.5 ml each of chloroform and water as described above. For assay systems with diethyl ether, 10% (v/v) diethyl ether is added to the substrate suspension or dispersion and the enzyme protein is added.

Lysophospholipase Activity The substrate (2/zmol) is dissolved in 0.3 ml of 0.2 M acetate buffer (pH 5.0) containing 2 mM EDTA and 0. I ml of enzyme protein. After incubation at 30° for 5 min, the reaction is stopped by adding 0.1 ml of 5%

[42]

PHOSPHOLIPASE B FROMPenicillium notatum

449

(w/v) bovine serum albumin and 0.4 ml of 10% perchloric acid on ice. The glycerophosphocholine liberated is determined as described above. Purification 13-17 Penicillium notatum (FIO 4640) is grown aerobically in a culture medium (pH 5.4) containing 3.5% corn steep liquor, 5.5% lactose, 0.7% KH2PO4, 0.5% CaCO3, 0.3% MgSO 4, and 0.25% soybean oil for 48 hr at 26 ° with continuous shaking in a 3 m 3 tank (2000 liters). The mycelia (51.2 kg) are obtained and stored at - 2 0 °. All purification steps thereafter are carried out at 4 ° unless otherwise stated. The mycelia are kindly supplied from Toyo Brewing Co., Ltd. One kilogram ofP. notatum cells is added to 5 volumes of 20 mM EDTA and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.0, homogenized with an Ultra Turrax disperser (Janke & Kunkel KG, FRG) set at maxin~um speed for 15 min and then centrifuged at 13,700 g for 25 min in the GS-3 rotor of a Sorvall centrifuge. The supernatant is brought to 90% saturation with solid ammonium sulfate and, after sitting overnight, is centrifuged at 13,700 g for 15 min. More than 90% of lysophospholipase activity, which is used as a measure ofphospholipase B activity throughout the purification, appears in the supernatant. The supernatant is concentrated by dialysis against 20 mM phosphate-1 mM EDTA buffer, pH 7.4, using a hollow fiber H1P10 type cartridge (Amicon, Danvers, MA). The dialyzate is applied to a DEAE-Sephadex A-50 column (2 × 11 cm) equilibrated with the same buffer. Inactive proteins are washed out with approximately 300 ml of 30 mM phosphate buffer at a flow rate of 15 ml/hr, and the enzyme is eluted with 150 ml of 80 mM phosphate buffer at the same flow rate as above. Then the pooled active fraction is dialyzed against 1 mM EDTA (pH 7) and freeze-dried. The preparation is stable for several months at - 20°. The post DEAE-Sephadex A-50 fraction is dissolved in 20 mM phosphate-1 mM EDTA buffer, pH 7.4, adjusted to pH 4.0 with glacial acetic acid, and applied to a phosphatidylserine-AH Sepharose column (1.3 × 5 cm, 1.6/~mol of phosphatidylserine bound/ml of gel) equilibrated with 0.2 M acetate-2 mM EDTA buffer, pH 4.0. The column is washed successively with the same buffer (60 ml), 0.2 M phosphate-2 mM EDTA buffer (pH 7.4,100 ml) and 0.2% Brij 58 in 20 mM phosphate-1 mM EDTA buffer z3 K. t4 N. 15 N. 16 T. ~7T.

Saito and K. Sato, Biochim. Biophys. Acta 151, 706 (1968). Kawasaki and K. Saito, Biochim. Biophys. Acta 296, 426 (1973). Kawasaki, J. Sugatani, and K. Saito, J. Biochem. (Tokyo) 77, 1233 (1975). Okumura, J. Sugatani, and K. Saito, Arch. Biochem. Biophys. 211, 419 (1981). Okumura, S. Kimura, and K. Saito, Biochim. Biophys. Acta 617, 264 (1980).

450

LYSOPHOSPHOLIPASE

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TABLE I PURIFICATION OF PHOSPHOLIPASE B FROM Penicillium notatum

Purification step

Total activitya (units/kg mycelium)

Original extract (NH4)2SO4 fractionation DEAE-Sephadex A-50 chromatography Phosphatidylserine-AH affinitychromatography Hydroxylapatitechromatography

57,000 52,000 41,000 34,000 23,000

Specific activity (units/mg protein) 1.5 125 381 5100 6900

Recovery (%) 100 91 72 60 41

a Enzyme activity is assayed using lysophosphatidylcholineas substrate. (pH 7.4, 50 ml) at a flow rate of 30 ml/hr. Then the enzyme is eluted with 0.1% Triton X-100 in 30 mM phosphate-1 mM EDTA buffer (pH 7.4, 50 ml). The affinity resin is prepared as follows: AH Sepharose 4B (Pharmacia, Piscataway, N J) is swollen and washed as recommended by the supplier. Phosphatidylserine (200 mg) purified from bovine brain and N,N'-dicyclohexylcarbodiimide (1.33 g) are added to 24 ml of the packed gel in 35 ml of tetrahydrofuran, and the pH is adjusted to 5.0. The coupling reaction is allowed to proceed for 24 hr at room temperature with gentle shaking after which acetic acid (2.5 ml) is added to block the uncoupled AH Sepharose. The gel is washed successively with tetrahydrofuran (90%, 200 ml), NaC1 (1 M, 500 ml), and is equilibrated and packed with the column buffer. The postphosphatidylserine-AH Sepharose fraction containing Triton X-100 is dialyzed against 5 mM phosphate-1 mM EDTA buffer, pH 7.4, and applied to hydroxylapatite column (2 × 9 cm) equilibrated with the same buffer. The column is washed with 8 mM phosphate-1 mM EDTA buffer, pH 7.4, and the enzyme is eluted with 40 mM phosphate-1 mM EDTA buffer, pH 7.4, at a flow rate of 30 ml/hr. The active fraction is dialyzed against 0.5 mM EDTA, pH 7.0, lyophilized, and stored at - 2 0 ° without a loss of activity for several months, but inactivated by freezing and thawing. The purification procedure is summarized in Table I. Characterization of Penicillium notaturn Phospholipase 12'16-18 The molecular size of phospholipase B (native form) is 95,000 Da in the presence and absence of 2-mercaptoethanol on slab sodium dodecyl is y. Takeuchi, T. Okumura, J. Sugatani, and K. Saito, Arch. Biochem. Biophys. 252, 206 (1987).

[42]

PHOSPHOLIPASE B FROMPenicillium notatum

/ 106K

95K ~

/

"=I

/

I=,

451

/

95K -.,--.- 78K

I•"

73K

32K

- SME

+ J~ME

FIG. 2. Slab SDS-PAGE patterns of the native and modified forms of phospholipase B in the presence or absence of 2-mercaptoethanol (/3ME).

sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). However, phospholipase B modified by protease(s) (modified form) gives a 106K band in the absence of 2-mercaptoethanol and is reductively cleaved to 78K, 73K, 37K, and 32K peptides by 2-mercaptoethanol. The protease(s), which attacks the phospholipase B molecule and is very sensitive to phenylmethylsulfonyl fluoride, exists intracellularly in P . n o t a t u m and increases at the late stationary phase of the growth. Figure 2 shows the typical pattern of the native and modified phospholipase B on slab SDS-PAGE in the presence and absence of 2-mercaptoethanol. The 37K plus 32K fragment and the 78K plus 73K fragment, respectively, are the N-terminal and C-terminal segments of the original 95K protein. The native and modified forms are glycoproteins with 29% carbohydrate (23% glucose and mannose and 6% N-acetylglucosamine) and have the same isoelectric point of 4.0. The circular dichroism spectrum in the far-ultraviolet region of the modified enzyme is different from that of the native one, showing the conformational change between the native and modified forms of the enzyme. The conformational change in protein structure ofphospholipase B results in a significant difference in pH dependence of enzyme activity (Fig. 3). The decrease in the B activity of the modified form is based on the decrease in both the A~ and A2 activities. Endoglycosidase H removes the carbohydrate from the native and modified enzymes to the same extent, but the B and lyso activities increase in

452

[42]

LYSOPHOSPHOLIPASE

4°°I

A

2o(:

lOG

3O

4.O

50 pH

e~o--

"~.o

2OOO

E

15oo

x o

I000

E

0

pH C

_>,4OOO

3~o

'

4"o

'

~o

pH

'

~

"

~o

[42]

PHOSPHOLIPASE B FROMPenicillium notatum

453

the modified enzyme and not in the native one. The carbohydrate moiety of phospholipase B may efficiently be involved in the enzymatic activity. Substrate Specificity Stereospecific .42 Activity and Nonstereospecific A t Activity. Penicillium notatum phospholipase B catalyzes the hydrolysis of the 2-acyl ester bond of L isomers of choline glycerophospholipids (1,2-diacyl-, l-O-alk1'-enyl-2-acyl-, and 1-O-alkyl-2-acyl-sn-glycerophosphocholines), but not that of the o isomers. The enzyme hydrolyzes both the 1-acyl ester bonds of the L and D isomers in a nonstereospecific phospholipase A1 activity. 19.20 Dipalmitoylglycerophospholipids with Different Polar Groups. The phospholipase B hydrolyzes phospholipids in the following order: phosphatidylserine > phosphatidylinositol > phosphatidic acid > phosphatidylcholine > phosphatidylethanolamine acid > cardiolipin. 21 Lysophospholipase Activity. The enzyme hydrolyzes 1-acyl-2-1yso-snglycero-3-phosphocholine more than the 2-acyl one. Lyso activity toward different fatty acyl chains shows the preference Cl0 < C12 > C14 > C16 ~ Cl8 .19,21

Lipase and Esterase Activities. Monoacyl(oleoyl or palmitoyl)glycerol is also hydrolyzed, especially in the presence of sodium taurocholate. But diacyl- and triacylglycerols including triacetoyl- and tributyroylglycerols, p-nitrophenyl acetate, and cholesteryl oleate are not hydrolyzed.19 A 1 and A 2 Activities o f the Phospholipase B. In conventional measurements with isotope-labeled 1,2-diacyl-sn-glycerophospholipids, A 1, A2, L1, and L 2 activities are indistinguishable. 19,2° But with 1,2-dipalmitoylsn-glycero-3-phosphocholine, 2,3-[14C]dipalmitoyl-sn-glycero-l-phospho choline, and 1-O-alkyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine, the B activity and its A 1 and A2 activities are determined independently. The system consists of a 5 mM substrate mixture as mentioned above, 0.2 M acetate buffer (pH 5.0) containing 2 mM EDTA, 30 mM Triton X-100, and the enzyme protein in a final volume of 0.4 ml. After incubation at 30° for 30 min, the reaction products are partitioned in chloroform and methat9 j. Sugatani, N. Kawasaki, and K. Saito, Biochim. Biophys. Acta 529, 29 (1978). 2oj. Sugatani, T. Okumura, and K. Saito, Biochim. Biophys. Acta 620, 372 (1980). el K. Saito and M. Kates, Biochim. Biophys. Acta 369, 245 (1974).

FI~. 3. pH dependence of activities of native and modified forms of phospholipase B. (A) Egg yolk 1,2-diacyl-sn-glycero-3-phosphocholine; (B) 1,2-dioctanoyl-sn-glycero-3-phosphocholine; (C) egg yolk 1-acyl-2-1yso-sn-glycero-3-phosphocholine.

454

LYSOPHOSPHOLIPASE

[42]

nol-water layers by the method of Bligh and Dyer. The lipid products and glycerophosphocholine liberated are analyzed as mentioned above.

Preparation o f Substrates 1-O-Alkyl-2-[14C]palmitoyl-sn-glycero-3-phosphocholine. The choline glycerophospholipid fraction is prepared from beef heart lipids by column chromatography on alumina and silicic acid and hydrolyzed with methanolic 0.25 M KOH at 37° for 30 min. 19The resulting 1-O-ack- 1'-enyl-2-1ysosn-glycero-3-phosphocholine (lysoplasmalogen) fraction is hydrogenated over platinum oxide, and mixed with [14C]palmitic acid (2/~mol) neutralized with tetraethylammonium hydroxide and palmitic anhydride (4/.Lmol) in vacuo at 80° overnight. The product is purified by column chromatography on silicic acid. 2,3-[14C]Dipalmitoyl-sn-glycero-1-phosphocholine. 2,3-Dipalmitoylsn-glycero-l-phosphocholine (40/xmol), obtained from hydrolysis of rac1,2-dipalmitoyl-sn-glycero-3-phosphocholine with phospholipase Az (Crotalus adamanteus), is incubated with lipase (Rhizopus arrhizus var. Delemar, 5 mg) in 10 ml of 19 mM sodium taurocholate, 5 mM CaClz, and 90 mM borate buffer (pH 5.7) at 37° for 3 hr. 2° The resulting monopalmitoylsn-glycero-l-phosphocholine is mixed with ['4C]palmitic acid neutralized with tetraethylammonium hydroxide and its fatty acid anhydride in vacuo at 80° overnight. Uniformly Labeled [14C,32p]diacylglycerophosphocholine. Candida lipolytica, NRRLY 1094, is grown on standard yeast medium in the presence of [14C] acetate and ortho[3Ep]phosphate. 21 The cells are harvested, and total lipids are extracted. The lipids are separated into individual components by preparative TLC. The labeled glycerophosphocholine and glycerophosphoethanolamine are chromatographically pure. The specific activity [disintegrations per minute (dpm)/nmol] of labeled glycerophosphocholine is 805 for 32p and 326 for 14C. The constituent fatty acids are 16 : 0 (7%), 16 : 1 (13%), 18 : 1 (36%), and 18 : 2 (44%); therefore, the suspension of this highly unsaturated substrate in acetate buffer (above) is found to be hydrolyzed in the absence of any activator and without being subjected to sonication (Fig. 1). l-[tgC]Palmitoyl-sn-glycero-3-phosphocholine and 2-[14C]Palmitoyl sn-glycero-3-phosphocholine. 1-[14C]Palmitoyl-sn-glycero-3-phosphocholine is prepared from hydrolysis of 1,2-[14C]palmitoyl-sn-glycero-3-phosphocholine with phospholipase A2 .19 2-[14C]Palmitoyl-sn-glycero-3-phosphocholine was prepared from 1-O-alk-l'-enyl-2-[14C]palmitoyl-snglycero-3-phosphocholine (2.5/xmol) dispersed in 1.5 ml petroleum ether and 1.5 ml of 0.1 M boric acid by the addition of iodine-saturated petroleum

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PHOSPHOLIPASE B FROMPenicillium notatum

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ether. These substrates should be used as soon as possible, because slow acyl migration occurs, particularly at acidic pH. 22 Egg Yolk Glycerophosphocholine and Its Lyso Compound. These substrates are prepared as previously reported) 3 Other Commercially Available Substrates. A homologous series of synthetic 1,2-diacyl-sn-glycero-3-phosphocholines containing two identical, saturated normal-chain fatty acids with even chain lengths from Ca to Cl8, as well as C18:1, and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine are purchased from Serdary Research Laboratories (London, Ontario, Canada). When necessary, the diacyl compounds are purified by preparative TLC. The short-chain glycerophosphocholines are purified on small column of silicic acid, eluted first with chloroform to remove simple lipids and then with a mixture of chloroform-methanol (1 : 1, v/v). 21 A homologous series of lysoglycerophosphocholines with acyl chains from C~4 to C18 is prepared from the corresponding diacylglycerophosphocholines by hydrolysis with phospholipase A 2 (Crotalus adamanteus) in an ether medium. 23 Lyso-C~0 and -C~2 glycerophosphocholine are purchased from Serdary Research Laboratories. All short-chain lysoglycerophosphocholines are unstable on storage in a mixture of chloroform-methanol (1 : 1, v/v) at 0° for longer than 2 weeks; therefore, they are always purified by silicic acid column immediately before use.

Stimulation and Inhibition Penicillium notatum phospholipase B activity is stimulated by diethyl ether, Triton X-100, and chlorpromazine (local anesthetic). Fe 2÷ and Fe 3÷ inhibit the B activity, but Mg 2÷ , Mn 2÷ , Zn 2÷ , Cu 2+ and Hg 2÷ do not. Ca 2+ is quite independent. Lyso activity is inhibited by detergents but not affected by the other reagents tested. Diisopropyl fluorophosphate (DFP) inhibits the B and lyso activities at a relatively high concentration (50 mM). Both activities are rather heat-labile) 5,19-21 Both activities are inhibited to the same extent by chemical modification of the enzyme protein with diethyl p-nitrophenyl phosphate [serine reagent, in 0.1 M NaC1 and 0.1 M acetate buffer (pH 5.5) in the presence of sodium taurocholate], N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide (tryptophan reagent, in the dark in 0.1 M acetate buffer, pH 4.0), and phenylglyoxal (arginine reagent, in the dark in 0.1 M N-ethylmorpholine-acetate buffer, pH 8.0). These observations suggest that both activities share the same active site of the phospholipase B. 22 A. Pliickthun and E. A. Dennis, Biochemistry 21, 1743 (1982). 23 D. J. Hanahan, M. Rodbell, and L. D. Turner, J. Biol. Chem. 2116, 431 (1954).

456

LYSOPHOSPHOLIPASE

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Conclusion Phospholipase B in P. notatum, at least, catalyzes the complete deacylation of all kinds of natural glycerophospholipids including lysoglycerophospholipids; therefore, the phospholipase B has both intrinsic B and lyso activities. Following proteolytic modification, the B activity is almost completely lost, but the lyso activity remains intact. The modified enzyme gives two large peptides and two small peptides by reductive cleavage with 2-mercaptoethanol. The large peptides are located at the C-terminal part, and the small peptides are at the N-terminal part of the native enzyme. In "Enzyme Nomenclature" published in 1984, phospholipase B is described as one of the other names for lysophospholipase (EC 3.1.1.5); however, as mentioned above, this may not be true for phospholipase B in P. notatum. How universally applicable the present model is to other lysophospholipases reported so far is the next problem to be solved. Acknowledgments This study is supported in part by a Grant-in-Aid for ScientificResearch (63480132)from the Ministry of Education, Science, and Culture of Japan, and by the Science Research Promotion Fund from the Japan Private School Promotion Foundation (1987-1989).

[43] L y s o p h o s p h o l i p a s e s I a n d II f r o m P 3 8 8 D 1 M a c r o p h a g e - l i k e Cell L i n e By YING YI ZHANG, RAYMOND A. DEEMS, and EDWARD A. DENNIS

Introduction 1-Acyl-sn-glycero-3-phosphorylcholine + H20 ~ glycero-3-phosphorylcholine + fatty acid

Until recently, membrane phospholipids were viewed as the inert building blocks of cellular membranes. We now appreciate that these "building blocks" are actively metabolized via a complex control system and that they dramatically affect numerous enzyme functions. In addition, it has been found that many of the products of phospholipid catabolism are also very potent cellular modulators. One of these products, lysophospholipid, is a strong detergent whose presence in the membrane can dramatically METHODS IN ENZYMOLOGY, VOL. 197

Copyright © 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

Phospholipase B from Penicillium notatum.

446 LYSOPHOSPHOLIPASE [42] [42] P h o s p h o l i p a s e B f r o m P e n i c i l l i u m n o t a t u m B y KUNIHIKO SAITO, JUNKO SUGATANI, and TA...
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